A tandem repeat in DNA represents two or more contiguous approximate copies of a pattern of nucleotides. Tandem repeats have been shown to be associated with a variety of human diseases. Dramatic expansion of trinucleotide repeats has been associated with such diseases as fragile-X mental retardation (see Verkerk, et al., (1991) Cell, 65, 905-914), Huntington's disease (see Huntington's Disease Collaborative Research Group. (1993) Cell, 72, 971-983), myotonic dystrophy (see Fu, et al., (1992) Science, 255, 1256-1258), spinal and bulbar muscular atrophy (see La Spada, et al., (1991) Nature, 352, 77-79) and Friedreich's ataxia (see Campuzano, et al., (1996) Science, 271, 1423-1427).
Fragile X syndrome (FXS, OMIM #300624) is the most common inherited mental retardation syndrome, affecting ˜1:4000 males and ˜1:8000 females. The disease is caused by the expansion of the trinucleotide CGG repeat in the 5′-untranslated (UTR) region of the fragile X mental retardation protein 1 (FMR1) gene. Methylation of the CGG tract leads to silencing of expression of the FMR1 gene. The American College of Medical Genetics (Maddalena et al. Genet Med 2001; 3(3):200-5; Sherman et al. Genet Med 2005; 7(8):584-7) defines a normal repeat length as between 5-44. Intermediate alleles of between 45-54 repeats show almost no expansion. Premutation alleles have 55-200 CGG, while full mutation alleles have >200 repeats. As an X-linked dominant disease, FXS is diagnosed more frequently in males than females, but females can present with milder symptoms, or with FXS due to skewed X-inactivation. Thirty-forty percent of male carriers of a premutation allele will suffer from Fragile X-associated Tremor and Ataxia (FXTAS) by age 50 (Hagerman et al. Ment Retard Dev Disabil Res Rev 2004; 10(1): 25-30; Hagerman et al. Am J Hum Genet 2004; 74(5): 1051-6) Various studies showed that the larger the repeat size the stronger the chance of presenting with FXTAS, but the penetrance i.e. the chance of being affected by FXTAS seems to be around 33% for male carriers of alleles ˜70 repeats or higher. Some female premutation carriers are affected with premature ovarian insufficiency (FXPOI; also know as premature ovarian failure) (Allingham-Hawkins et al. Am J Med Genet 1999; 83(4):322-5; Hundscheid et al. Hum. Reprod. 2001; 13(3): 457-462). Female premutation carriers showed overall higher levels of follicular stimulating hormone than normal or full mutation carriers and decreased levels of anti-Müllerian hormone, Inhibin A and inhibin B, all indicators of ovarian decline (Hundscheid et al. Hum. Reprod. 2001; 13(3): 457-462). The reported penetrance i.e. the chance of being affected by FXPOI in female premutation carriers is 20-28% of larger alleles.
Several studies have examined the carrier frequency of premutation and/or full mutation alleles in FMR1 gene in different populations, and as in any other genetic disease, the frequency varies by population and region; 1:113 in Israel (Toledano-Alhadef et al. Am J Hum Genet 2001; 69(2):351-60), 1:259 in Quebec (Rousseau et al. Am J Hum Genet 1995; 57(5):1006-18), in Finland the frequency was 1:246, and in the U.S. state of Georgia it was 1:436. The frequency was found to be between 1:257-1:382 in the U.S. population in general (Cronister et al. Genet Med 2005; 7(4):246-50). Methods of amplifying the CGG repeat tract in the FMR1 gene has previously been reported. See, Fu et al. Cell 1991; 67: 1047-1058; Erster et al. Hum Genet. 1992; 90: 55-61; Pergolizzi et al. Lancet. 1992; 339: 271-272; Warner et al. J Med Genet. 1996; 33: 1022-1026; Daniels et al. J Assist Reprod Genet. 1996; 13(2): 163-169; Strelnikov et al. Hum Mutat. 1999; 13(2): 166-169; Zhou et al. J Med Genet. 2004; 41(4): e45; Saluto et al. J Mol Diagn. 2005; 7(5): 605-612; Zhou et al. Clin Chem. 2006; 52(8): 1492-1500; Dahl et al. Clin Chem. 2007; 53(4): 790-793; Strom et al. Genet Med. 2007; 9(4): 199-207; Khaniani et al. Mol Cytogenet. April 8; 1:5; Tassone et al. J Mol Diagn. 2008; 10(1): 43-49; Filipovic-Sadic et al. Clin Chem. Jan. 7 2010 (epub); Lyon et al. J Mol. Diagn. 2010; 12(4): 505-511; Hantash et al. Genet Med. Feb. 17 2010 (epub); U.S. Pat. Nos. 5,658,764, 6,200,747, and 6,114,150, and US Patent Application Publication 2008/0113355.
Fragile X E (FRAXE) mental retardation (OMIM 309548) is associated to a fragile site localized in chromosome Xq28 and is the cause of a non-syndromic X-linked mental retardation affecting 1/50,000 newborn males. The disorder is due to the silencing of the Fragile X Mental Retardation 2 (FMR2) gene, as a consequence of a CCG expansion located upstream to this gene. FRAXE alleles can be divided into four categories: normal (6-30 CCG repeats), intermediate (31-60 CCG repeats), premutation (61-200 CCG repeats), and full mutation (over 200 CCG repeats). See, Gecz et al. Nat. Genet. 1996; 13: 105-108; Gu et al. Nat. Genet. 1996; 13:109-113; Knight et al. Cell. 1993; 74:127-134; and Strelnikov et al. Hum Mutat. 1999; 13(2):166-9.